DE3923279C2 - - Google Patents
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- Publication number
- DE3923279C2 DE3923279C2 DE3923279A DE3923279A DE3923279C2 DE 3923279 C2 DE3923279 C2 DE 3923279C2 DE 3923279 A DE3923279 A DE 3923279A DE 3923279 A DE3923279 A DE 3923279A DE 3923279 C2 DE3923279 C2 DE 3923279C2
- Authority
- DE
- Germany
- Prior art keywords
- cell
- cells
- carrier
- carriers
- cultivation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/02—Membranes; Filters
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/44—Multiple separable units; Modules
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/14—Scaffolds; Matrices
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/10—Perfusion
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- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Sustainable Development (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Clinical Laboratory Science (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Description
Es ist allgemeines Wissensgut, daß die Zusammensetzung der extrazellulären Biomatrix bei der Zellkultivation in biologischen und biotechnologischen Experimenten von sehr großer Bedeutung für das Anhaften und die spezielle Differenzierungsleistung der jeweiligen Zellen ist. Eine solche extrazelluläre Matrix besteht unter anderem aus verschiedenen Kollagenen, Laminin, Fibronektin und anderen kollagen- oder nichtkollagenartigen Proteinen. In vielen Experimenten hat sich gezeigt, daß hochspezialisierte Zellen ohne ein solches Trägermaterial nicht kultiviert werden können.It is common knowledge that the composition of the extracellular biomatrix in cell cultivation in biological and biotechnological experiments of great importance for the attachment and the special differentiation of the respective cells. Such an extracellular matrix exists among other things from different collagens, laminin, fibronectin and other collagen or non-collagenous proteins. In Many experiments have shown that highly specialized Cells cannot be cultivated without such a carrier material can.
Neben der Mikrocarriertechnik gibt es bisher nur wenige Hilfsmittel, die es ermöglichen, lebende Zellen auf Supporten zu kultivieren. Dabei handelt es sich um Hohlzylinder mit einem auf einer Seite aufgebrachten Filtermaterial, auf dem die Zellen wachsen können. Es ist weiterhin bekannt, daß eine zusammenhängende Zellschicht auf einem porösen oder halbdurchlässigen Substrat kultiviert werden kann, wobei das Substrat in scheibenförmiger Form in eine ringförmige Halterung zu spannen ist, die so stabilisiert in das entsprechende Kulturgefäß eingebracht wird (US-Patentschrift 46 08 342).In addition to microcarrier technology, there are currently only a few Tools that enable living cells to be supported cultivate. These are hollow cylinders with a one side of filter material on which the cells can grow. It is also known that a contiguous cell layer on a porous or semi-permeable substrate can be grown, the Disc-shaped substrate in an annular holder is to be tensioned, which is stabilized in the corresponding Culture vessel is introduced (US Patent 46 08 342).
Aufgabe der Erfindung ist es, eine Vorrichtung für die Kultivierung von Zellen zu entwickeln, in der Zellen unter möglichst natürlichen Bedingungen gehalten werden können und bei der das kultivierte Material von einer Versuchsanordnung schnell in eine andere überführt werden kann. Die Zellen wachsen dabei in einem speziellen Kulturgefäß mit Ein- und Ausstromöffnung für das Kulturmedium auf einzelnen (Fig. 1) oder mehreren (Fig. 2) austauschbaren, blatt- oder folienartigen Zellträgern. Diese Technik erlaubt die Kultivierung von hoch differenzierten Zellen im kleinen Labormaßstab, wie durch beliebige Vergrößerung der Träger in immer größerer Dimension wie z. B. in Bioreaktoren. Die Erfindung betrifft die im Anspruch 1 beschriebene Vorrichtung. Weitere Ausgestaltungen sind aus den Ansprüchen 2 bis 7 zu entnehmen. The object of the invention is to develop a device for the cultivation of cells, in which cells can be kept under conditions that are as natural as possible and in which the cultivated material can be quickly transferred from one test arrangement to another. The cells grow in a special culture vessel with an inflow and outflow opening for the culture medium on individual ( FIG. 1) or several ( FIG. 2) exchangeable, sheet-like or film-like cell carriers. This technique allows the cultivation of highly differentiated cells on a small laboratory scale, as by any enlargement of the carrier in ever larger dimensions such as. B. in bioreactors. The invention relates to the device described in claim 1. Further refinements can be found in claims 2 to 7.
Durch Überführung von kultivierten Zellen aus einfachen Kulturschalen in die spezielle Vorrichtung (Fig. 1) wird eine neue Art der Zellkultivation unter Perfusionsbedingungen mit apikalen und basalen Kulturmediumgradienten und damit z. B. für Epithelzellen unter nahezu natürlichen Bedingungen möglich. Dies wird unter anderem dadurch erreicht, daß die lebenden Zellen nicht fest mit dem impermeablen Boden eines normalen Kulturgefäßes verbunden sind, sondern sich auf einem in die Vorrichtung einzulegenden, also den Kulturschalenboden in der Qualität verbessernden und damit den natürlichen Bedingungen angleichenden folienartigen und permeablen Träger von variabler Größe befindet, der jederzeit wieder aus der Vorrichtung entfernt werden kann.By transferring cultured cells from simple culture dishes into the special device ( Fig. 1), a new type of cell cultivation under perfusion conditions with apical and basal culture medium gradients and thus z. B. possible for epithelial cells under almost natural conditions. This is achieved, among other things, by the fact that the living cells are not firmly connected to the impermeable bottom of a normal culture vessel, but instead are placed on a film-like and permeable carrier which is to be inserted into the device, that is to say the quality of the culture dish bottom and thus conforming to the natural conditions variable size, which can be removed from the device at any time.
Durch Ausschneiden oder Ausstanzen einer geeigneten Folie, eines Filters, Gitters oder sonstigen geeigneten Trägermaterials sowie eventueller Beschichtung in jeder Größe und Form z. B. mit einem zellulären oder extrazellulären Matrixprodukt und durch Verankerung dieses Trägers in einer flachen Halterung wird der Zellträger hergestellt.By cutting or punching out a suitable foil, one Filters, grids or other suitable carrier material as well possible coating in any size and shape z. B. with a cellular or extracellular matrix product and by Anchoring this carrier in a flat bracket is the Cell carrier made.
Das ausgestanzte Trägermaterial wird dann in einen Ring eingelegt. Mit einem kleineren, eng sitzenden flachen Ring wird das Trägermaterial im größeren Ring fixiert, ähnlich einem Trommelfell auf einer Trommel. Nachdem man den kleinen Ring eingepreßt hat, ist die Befestigungsprozedur beendet. Wenn eine Beschichtung notwendig werden sollte, kann dies entweder vor dem Montieren des Zellträgers oder aber auch danach geschehen.The punched carrier material is then in a ring inserted. With a smaller, tight-fitting flat ring the carrier material fixed in the larger ring, similar to one Eardrum on a drum. After getting the little ring has pressed in, the fastening procedure is finished. When a Coating should be necessary, this can be done either before Assemble the cell support or do it afterwards.
Es ist ein wesentliches Kriterium der Zellträger, daß sie als blatt- oder folienartige Unterlagen in die Vorrichtung für die Kultivierung von Zellen eingelegt werden, um dort lebenden Zellen eine gute Anhaftung und eine möglichst hohe Differenzierung zu erleichtern oder unter möglichst natürlichen Bedingungen überhaupt erst zu ermöglichen. Die Zellträger verbessern somit für lebende Zellen entscheidend die Qualität der sehr unspezifischen Charakteristika von sonstigen Kulturgefäßen. It is an essential criterion of the cell carrier that it is considered sheet or foil-like documents in the device for the Cultivation of cells can be inserted to cells living there good adhesion and the greatest possible differentiation facilitate or under as natural conditions as possible to make it possible at all. The cell carriers thus improve for living cells the quality of the very crucial unspecific characteristics of other culture vessels.
Perfusionssystem: Die erfindungsgemäße Vorrichtung für die Kultivierung von Zellen besitzt apikal und basal eine Einstrom- und Ausstromöffnung (Fig. 1). Durch eine seitliche Abdichtung des folienartigen Zellträgers, die den Innenraum der Vorrichtung in zwei voneinander getrennte Räume aufteilt, wird eine getrennte apikale und basale Perfusion der Zellen parallel zu einer elektrophysiologischen Registrierung von Transportvorgängen bei geringstem Totraumvolumen ermöglicht. Diese seitliche Abdichtung des Zellträgers in der speziellen Vorrichtung zur Kultivierung von Zellen kann sehr leicht ein Leck verhindern. Dadurch wird es möglich, Transportvorgänge zwischen der apikalen und basalen Seite zu messen. Hinzu kommt, daß durch eine individuelle apikale und basale Perfusion mit hypo- oder hypertonen Medien die Bildung eines Gradienten während der Kultur oder des Experiments ermöglicht wird, wie das z. B. in der Niere vorkommt. Diese Art der Nutzung kultivierter Zellen ist einzigartig, öffnet ein neues Feld in der zellbiologischen Wissenschaft und war bisher in dieser Art nicht möglich.Perfusion system: The device according to the invention for the cultivation of cells has an inflow and outflow opening apically and basally ( FIG. 1). A lateral sealing of the film-like cell carrier, which divides the interior of the device into two separate spaces, enables separate apical and basal perfusion of the cells in parallel with an electrophysiological registration of transport processes with the smallest dead space volume. This lateral sealing of the cell carrier in the special device for cultivating cells can very easily prevent a leak. This makes it possible to measure transport processes between the apical and basal side. In addition, the formation of a gradient during the culture or experiment is made possible by an individual apical and basal perfusion with hypo- or hypertonic media. B. occurs in the kidney. This type of use of cultivated cells is unique, opens up a new field in cell biological science and was previously not possible in this way.
Bioreaktoren: In einer besonderen Ausführungsform der erfindungsgemäßen Vorrichtung werden mehrere Zellträger gestapelt (Fig. 2). Dabei kann sowohl der Durchmesser der Träger in der Vorrichtung variieren, es kann aber auch die Stapelhöhe beliebig gewählt werden.Bioreactors: In a special embodiment of the device according to the invention, several cell carriers are stacked ( FIG. 2). Both the diameter of the carrier in the device can vary, but the stacking height can also be chosen as desired.
In der speziellen Vorrichtung können die Träger perfundiert werden, nämlich von der apikalen und von der basalen Seite mit einem extrem niedrigen Totraumvolumen.The carriers can be perfused in the special device with the apical and the basal side an extremely low dead space volume.
In der Vorrichtung können entweder Träger mit gleichartigen Zellen eingebracht werden, oder es können auch mit unterschiedlichen Zellarten bewachsene Träger zur Anwendung kommen, wodurch unter Umständen eine positive Beeinflussung der Zellen untereinander ermöglicht wird.In the device, either carriers with similar Cells can be introduced, or it can also be used carriers overgrown with different cell types for use come, which may have a positive influence on the Cells with each other is made possible.
Der Vorteil der erfindungsgemäßen Vorrichtung ist darin zu sehen, daß jeder einzelne Zellträger für sich allein in der Vorrichtung kontrolliert werden kann. Dies geschieht dadurch, daß jeder Träger von apikal und basal eine Flüssigkeitszufuhr und Flüssigkeitsabfuhr besitzt (Fig. 2). Damit kann z. B. jeder Zellträger für sich allein auf eine Infektion oder Sekretion hin kontrolliert werden. Das Reaktorrohr der Vorrichtung kann leicht geöffnet werden und jeder gewünschte Träger kann für sich allein herausgenommen und ersetzt werden.The advantage of the device according to the invention can be seen in the fact that each individual cell carrier can be checked individually in the device. This is done in that each apical and basal carrier has a fluid supply and drainage ( Fig. 2). So z. B. each cell carrier can be checked individually for infection or secretion. The reactor tube of the device can be opened easily and any desired support can be taken out and replaced on its own.
Fig. 1:
Schematischer Längsschnitt einer erfindungsgemäßen Vorrichtung
zur Kultivierung von Zellen (1) auf einzelnen Zellträgern (2)
mit einer apikalen (3) und basalen (4) Flüssigkeitszufuhr und
Flüssigkeitsabfuhr. Fig. 1:
Schematic longitudinal section of a device according to the invention for the cultivation of cells ( 1 ) on individual cell supports ( 2 ) with an apical ( 3 ) and basal ( 4 ) fluid supply and drainage.
Fig. 2:
Schematischer Längsschnitt einer erfindungsgemäßen Vorrichtung
zur Kultivierung von Zellen (1) auf mehreren Zellträgern (2)
mit Einstromöffnung (3) und Ausstromöffnung (4) für das
Kulturmedium. (5) Abdichtung der Reaktorsegmente. Fig. 2:
Schematic longitudinal section of a device according to the invention for the cultivation of cells ( 1 ) on several cell supports ( 2 ) with inflow opening ( 3 ) and outflow opening ( 4 ) for the culture medium. ( 5 ) Sealing the reactor segments.
Claims (7)
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
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DE3923279A DE3923279A1 (en) | 1989-07-14 | 1989-07-14 | MINUSHEETS IS A NEW PRODUCT FOR CULTIVATING CELLS IN ANY CONTAINERS IN A HIGHLY DIFFERENTIATED FORM ON THE MOST NATURAL DOCUMENT |
US07/627,729 US5190878A (en) | 1989-07-14 | 1990-12-14 | Apparatus for cultivating cells |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE3923279A DE3923279A1 (en) | 1989-07-14 | 1989-07-14 | MINUSHEETS IS A NEW PRODUCT FOR CULTIVATING CELLS IN ANY CONTAINERS IN A HIGHLY DIFFERENTIATED FORM ON THE MOST NATURAL DOCUMENT |
Publications (2)
Publication Number | Publication Date |
---|---|
DE3923279A1 DE3923279A1 (en) | 1990-01-18 |
DE3923279C2 true DE3923279C2 (en) | 1991-01-31 |
Family
ID=6385013
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
DE3923279A Granted DE3923279A1 (en) | 1989-07-14 | 1989-07-14 | MINUSHEETS IS A NEW PRODUCT FOR CULTIVATING CELLS IN ANY CONTAINERS IN A HIGHLY DIFFERENTIATED FORM ON THE MOST NATURAL DOCUMENT |
Country Status (2)
Country | Link |
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US (1) | US5190878A (en) |
DE (1) | DE3923279A1 (en) |
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-
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DE19808055A1 (en) * | 1998-02-27 | 1999-09-02 | Adamietz | Tissue engineering procedure extracting and culturing cells with mechanical stimulation to simulate in-vivo growth |
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Also Published As
Publication number | Publication date |
---|---|
US5190878A (en) | 1993-03-02 |
DE3923279A1 (en) | 1990-01-18 |
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